The resting behavior of malaria vectors in different ecological zones of Ghana and its implications for vector control

Background In sub-Saharan Africa there is widespread use of long-lasting insecticidal nets and indoor residual spraying to help control the densities of malaria vectors and decrease the incidence of malaria. This study was carried out to investigate the resting behavior, host preference and infection with Plasmodium falciparum of malaria vectors in Ghana in the context of the increasing insecticide resistance of malaria vectors in sub-Saharan Africa. Methods Indoor and outdoor resting anopheline mosquitoes were sampled during the dry and rainy seasons in five sites in three ecological zones [Sahel savannah (Kpalsogo, Pagaza, Libga); coastal savannah (Anyakpor); and forest (Konongo)]. Polymerase chain reaction-based molecular diagnostics were used to determine speciation, genotypes for knockdown resistance mutations (L1014S and L1014F) and the G119S ace1 mutation, specific host blood meal origins and sporozoite infection in the field-collected mosquitoes. Results Anopheles gambiae sensu lato (s.l.) predominated (89.95%, n = 1718), followed by Anopheles rufipes (8.48%, n = 162) and Anopheles funestus s.l. (1.57%, n = 30). Sibling species of the Anopheles gambiae s.l. revealed Anopheles coluzzii accounted for 63% (95% confidence interval = 57.10–68.91) and 27% (95% confidence interval = 21.66–32.55) was Anopheles gambiae s. s.. The mean resting density of An. gambiae s.l. was higher outdoors (79.63%; 1368/1718) than indoors (20.37%; 350/1718) (Wilcoxon rank sum test, Z = − 4.815, P < 0.0001). The kdr west L1014F and the ace1 mutation frequencies were higher in indoor resting An. coluzzii and An. gambiae in the Sahel savannah sites than in the forest and coastal savannah sites. Overall, the blood meal analyses revealed that a larger proportion of the malaria vectors preferred feeding on humans (70.2%) than on animals (29.8%) in all of the sites. Sporozoites were only detected in indoor resting An. coluzzii from the Sahel savannah (5.0%) and forest (2.5%) zones. Conclusions This study reports high outdoor resting densities of An. gambiae and An. coluzzii with high kdr west mutation frequencies, and the presence of malaria vectors indoors despite the use of long-lasting insecticidal nets and indoor residual spraying. Continuous monitoring of changes in the resting behavior of mosquitoes and the implementation of complementary malaria control interventions that target outdoor resting Anopheles mosquitoes are necessary in Ghana. Graphical abstract


Background
Malaria is a major public health problem in Africa, and was responsible for an estimated 241 million episodes and 624,000 deaths worldwide in 2020 [1]. In Ghana, malaria is responsible for more than 5.5 million infections and 37 deaths per 1000 population [2,3] despite tremendous efforts to scale-up vector control interventions there, particularly in the use of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) [4,5]. These anti-vector interventions led to a remarkable reduction in vector populations [6][7][8][9] and malaria transmission [10,11] in the past. However, there are reports of a resurgence in malaria in many parts of sub-Saharan Africa [12,13]. Some of the challenges associated with this resurgence include the emergence of insecticide resistance, behavioral modifications (shifts in the biting and resting behavior of vectors, from indoors to outdoors) and a shift in host species preference from humans to animals [7,[14][15][16].
These challenges have arisen as a consequence of adaptations of malaria vectors to the high use of insecticides for their control [17]. For instance, following the introduction of LLINs, there have been reports of a shift in the biting behavior of Anopheles gambiae and Anopheles funestus in Kenya [7,18] and Anopheles funestus in Benin and Senegal [19,20]. Whilst the long-term use of LLINs has increased the proportions of Anopheles gambiae and Anopheles melas feeding outdoors in Equatorial Guinea [14], in Tanzania, the long-term use of LLINs was reported to be associated with shifts in the outdoor resting rates of Anopheles gambiae, Anopheles arabiensis and Anopheles funestus [15,21]. These behavioral changes, however, are not consistent, with some countries reporting high indoor resting densities of An. gambiae and An. funestus despite the long-term use of LLINs and IRS [22][23][24].
The widespread insecticide resistance of malaria vector populations in Africa is a major threat to current malaria control programmes there. Studies from Côte d'Ivoire [25], Togo [26,27], Benin [28], Burkina Faso [29,30], Cameroon [31,32] and Kenya [33,34] have reported high metabolic resistance and target site modifications in malaria vectors with respect to insecticides used against them. The acetylcholinesterase (ace1) target site mutation G119S, which enables resistance to organophosphates and carbamates, and the voltage-gated sodium channel knockdown resistance gene (kdr), which plays a major role in resistance to pyrethroids, are the most common and important target site mechanisms of mosquito vectors in Ghana [35][36][37].
The primary malaria vectors in Ghana are members of the Anopheles gambiae sensu lato (s.l.) species complex [Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, Anopheles coluzzii and Anopheles melas] and Anopheles funestus s.s. [38][39][40][41]. In view of the resurgence of malaria transmission in Africa [42,43], which is of increasing concern, there is a need to improve control intervention strategies through a better understanding of vector resting and feeding behavior in different settings and under varying seasonal patterns. This is crucial for the success of current vector control tools, and could provide a guide for improved efforts for the control of malaria in endemic regions.
The objective of this study was to investigate the resting behavior, species composition, insecticide resistance status and Plasmodium falciparum infections of malaria vectors in three ecological zones of Ghana (the coastal savannah zone in the south, the forest zone in the center, and the Sahel savannah zone in the north of the country). These ecological zones have varying climatic and other environmental conditions that are suitable for the propagation of Anopheles mosquitoes and malaria parasites [44]. The coastal savannah and forest zones have a bimodal rainfall pattern, allowing for two peaks of malaria transmission, while the Sahel savannah zone has a unimodal rainfall pattern leading to seasonal malaria transmission. The results of this study may improve our understanding of the impacts of current malaria control programmes on malaria vector populations, and their effects on mosquito resting behavior.

Study sites
This study was carried out in five sites in three ecological zones of Ghana: Anyakpor in the coastal savannah zone; Dwease in the forest zone; and Kpalsogou, Libga and Pagaza in the Sahel savannah zone (Fig. 1).
Anyakpor (5°46′51.96"N, 0°35′12.84"E) is a village in the coastal savannah zone, about 5 km west of Ada Foah in southern Ghana. The coastal savannah has a tropical savannah climate, with average annual precipitation of 787 mm. Dwease (6°32′3.05"N, 1°14′42.22"W) is a village near Konongo, in the Asante-Akim Central District in the middle of Ghana, and is located in the forest zone. The forest zone has a tropical rainforest climate, with average annual precipitation of 1399.5 mm. Both the coastal savannah and forest zones generally have a bimodal pattern of rainfall, with the long rainy season from March to June and the short rainy season from October to November; their mean annual temperature is 26.5 °C. The sites in the Sahel savannah ecological zone were Kpalsogou (9°33′45.2"N, 1°01′54.6"W), a village in Kumbungu District in the northern region; Pagaza (9°22′33.34"N, 0°42′29.67"W) in the Tamale metropolitan area; and Libga (9°35′32.26"N, 0°50′48.8"W), a village in the Savelugu-Nanton Municipal District. These sites have a unimodal rainfall pattern from May to November and a mean annual temperature of 28 °C (the maximum recorded temperature is presently 42 °C).

Mosquito collection
Mosquitoes were sampled in May 2019 during the rainy season at the sites in the coastal savannah and forest zones (Anyakpor and Konongo, respectively), in September 2019 at the sites in the Sahel savannah zone (Pagaza, Libga, and Kpalsogou), and in the dry season from February to March 2019 at all the study sites. Sixteen houses were randomly selected in each study site, and four of these houses were sampled for mosquitoes on each sampling night. Sampling was done over 4 days during both the dry and rainy seasons. The global positioning system coordinates of each site were determined and recorded.
Malaria vectors resting indoors were sampled from 0500 to 0700 hours using pyrethrum spray catches [45]. A Prokopack aspirator (John W. Hock, Gainesville, FL) was used from 0500 to 0700 hours to collect mosquitoes resting indoors or outdoors [46]. For indoor collection, mosquitoes resting on walls, under roofs, on ceilings, and under beds were systematically aspirated. Outdoor sampling points included kitchens, granaries, places where animals rested and locations where humans rested in the evening. Additionally, outdoor resting mosquitoes were collected from pit shelters constructed 10 m from selected houses according to Muirhead-Thomson's method [47]. Resting mosquitoes were collected from 0600 to 0700 hours from cavities in the pit shelters by using hand-held mouth aspirators.

Morphological identification
All of the mosquitoes caught were counted and anopheline mosquitoes were sorted morphologically according to the identification keys of Gillies and Coetzee [48]. Sampled mosquitoes were further classified according to abdominal status as unfed, freshly fed, half-gravid and gravid. Mosquitoes collected using each collection method were stored in separately labeled vials in 95% ethanol. Samples were stored at the insectary of

Sibling species discrimination
Sibling species of the An. gambiae s.l. species complex were distinguished using the protocols of Scott et al. [49] and Fanello et al. [50]. Template DNA was extracted from one leg of each mosquito and placed directly into the polymerase chain reaction (PCR) Master Mix for amplification.

Detection of sporozoites
The head and thorax of mosquitoes in pooled samples (10 mosquitoes in each pool) were used to detect the presence of P. falciparum sporozoites by PCR as described by Echeverry et al. [51].

Detection of blood meal sources
The abdomens of the blood-fed Anopheles mosquitoes were cut into transverse sections. Genomic DNA was extracted from the mosquito abdomens using the ZR DNA MicroPrep kit (Zymo Research, CA) following the manufacturer's instructions. One universal reverse primer and five animal-specific (human, cow, goat, pig, and dog) forward primers were used for amplification of the mitochondrial cytochrome b gene to test for specific host blood meal origin using conventional PCR [52]. Positive controls were included for each host in the PCR analyses, and laboratory-reared unfed An. gambiae were used as the negative control.

Genotyping for kdr and ace1 mutations
For kdr mutation genotyping, DNA was extracted from mosquito legs using the ZR DNA MicroPrep kit (Zymo Research) following the manufacturer's instructions. Standard PCR assays for the L1014F kdr allele were used to test for the presence of the kdr gene using a modification of the protocol described by Ahadji-Dabla et al. [53]. The G119S mutation of the ace1 gene was assessed using the PCR protocol described by Weill et al. [54].

Data analysis
Densities of resting anopheline mosquitoes were calculated as the number of female mosquitoes/trap per night for each trapping method. The Mann-Whitney U-test was used to compare malaria vector density between indoor and outdoor locations. The chi-square test was used to examine the differences in seasonal abundance and malaria vector species composition between resting locations (indoors and outdoors).
Human blood index (HBI) was calculated as the proportion of blood-fed mosquito samples that had fed on humans relative to the total tested for blood meal origin. The sporozoite infection rate, expressed as the proportion of mosquitoes positive for Plasmodium sporozoites, was calculated by dividing the number of sporozoitepositive mosquitoes by the total number of mosquitoes assayed.
The kdr L1014F and ace1 G119S mutation frequencies were calculated according to the following formula [53]: Sporozoite infection rates were calculated for the pooled samples (10 samples per pool) of mosquitoes by using the formula given by Gu [55]: The maximum likelihood estimate (MLE) of the pooled mosquitoes was determined using the frequentist MLE model in R [56]. In the Sahel savannah zone, the percentage of An. coluzzii was higher in the dry season (81% and 91%) than in the rainy season (67% and 61%) for Kpalsogou and Pagaza, respectively (Fig. 2). In contrast, the percentage of An. gambiae was higher in the dry season (60%) than in the rainy season (50%) in Ligba. In Anyakpor and Konongo, higher densities of An. coluzzii were detected in the dry season (94% and 64%, respectively) compared to the rainy season (66% and 60%, respectively).

kdr resistance mutations of indoor resting compared to outdoor resting An. gambiae
A total of 538 An. gambiae s.l. samples were genotyped for the presence of the L1014S, L1014F and G119S ace1 mutations. The L1014F allele of kdr was identified in 100% (538) of the samples, with the majority of mosquitoes being homozygous for the kdr allele (70.6%; 380/538). Overall, there was little difference between the kdr mutation frequencies of mosquitoes collected indoors and outdoors for all the study sites (Table 4). Furthermore, the kdr L1014S mutation was not detected in this study.

G119S ace1 mutations in indoor and outdoor resting malaria vectors
The G119S ace1 mutation was detected in 79.9% (215/538) of the mosquitoes tested (Table 5). All the mosquitoes with the resistant allele were heterozygous for this mutation. Overall, similar ace1 mutation frequencies were detected for indoor and outdoor resting mosquitoes in the Sahel savannah zone (0.8 vs. 0.8). In the forest zone, the frequency of ace1 mutations was slightly higher in mosquitoes resting indoors (0.9) than in those resting outdoors (0.8), but was higher in mosquitoes resting outdoors (1) Table 5). In Kpalsogou and Pagaza (Sahel savannah sites), the ace1 mutation frequency was higher for An. gambiae resting indoors (0.5 and 0.5, respectively) than outdoors (0.4 and 0.4, respectively).

Blood meal sources
The 362 An. gambiae s.l. blood-fed mosquito specimens analyzed for blood meal origins had a HBI of 70

Discussion
Behavioral diversification in vector populations in areas with widespread use of LLINs and IRS is a threat to the efficacy of vector control strategies [7,8,[14][15][16]57]. This study investigated the behavior of malaria vectors, specifically their resting and feeding choices, and their rates of infection with Plasmodium falciparum sporozoites, in the context of increasing insecticide resistance. Overall, this study revealed high outdoor resting densities of malaria vectors, but that these mosquitoes had lower sporozoite infection rates than those resting indoors. More mosquitoes with insecticide resistance mutations were found resting indoors than outdoors at the different sites in the three ecological zones of Ghana included in this study. The use of LLINs and IRS in Kpalsogou, Libga and Pagaza (Sahel savannah zone) is driving more malaria vectors to rest outdoors, as observed for An. coluzzii and An. gambiae collected from different sites in a previous study in Ghana [58]. This could lead to an increase in malaria transmission outdoors since these main malaria vectors can bite unprotected humans outdoors and also rest outdoors to avoid contact with the insecticides that are used indoors [59,60]. This behavioral change in malaria vector populations is detrimental to the efficacy of LLINS and IRS vector control strategies as these mainly target vectors resting indoors. A tendency for the malaria vectors (An. coluzzii and An. gambiae) to rest and feed outdoors due to the long-term use of LLINs has been reported from Equatorial Guinea [14] and Tanzania [15,21].
Generally, more mosquitoes were collected during the rainy season than the dry season, apart from in Kpalsogou and Konongo. Kpalsogou has a dam for irrigation, and breeding habitats are created when the water held in the dam flows through the conduits to the farmland that it feeds. In Konongo, breeding habitats are found along a stream that flows through the village. However, both the dam and the stream flood during the rainy season, and mosquito larvae are washed away as a result. This might explain the low vector abundance found in Konongo and Kpalsogou during the rainy season.
One interesting observation was that, the frequencies of the kdr west mutation L1014F and the ace1 mutation were higher in indoor resting An. coluzzii and An. gambiae in the Sahel savannah sites compared to the forest and coastal savannah sites. This may be because the increase in IRS and LLINs use in the Sahel savannah sites has selected for malaria vectors with kdr and ace1 mutations. In addition, a behavioral change in populations of malaria vectors was observed in previous studies [31,[67][68][69], whereby those with low kdr mutation frequencies rested outdoors and thus avoided contact with the insecticides that were used indoors. Previous studies from Ghana [35,36] have reported similar frequencies of kdr L1014F in An. coluzzii to that found in the present study. Similar to other studies carried out in Ghana [35,    36], no kdr east allele was detected in the present study. However, kdr east allele 1014S has been reported in An. coluzzii, An. gambiae, and An. arabiensis from Burkina Faso [61], and in both An. coluzzii and An. gambiae from Togo [27].
The blood meal analyses revealed that a large proportion of the malaria vectors preferred feeding on humans than on animals in almost all of the sites. This preference for human hosts and the higher proportions of outdoor resting An. gambiae and An. coluzzii are of great concern for malaria elimination efforts due the efficacy of these mosquitoes in transmitting malaria. A similar study to ours carried out by Orsborne et al. [62] in the coastal area of Ghana likewise reported that blood-fed mosquitoes caught indoors had higher HBI and a lower bovine blood index than those caught outdoors. Sporozoite infections were only found in indoor resting malaria vectors, collected during the wet season from Kpalsogou and during the dry season from Konongo. This indicates that malaria transmission may occur more indoors than outdoors [63]. However, in comparison to the results of the present study, higher sporozoite infections were found in An. gambiae and An. coluzzii resting outdoors in Kenya and Burkina Faso [22,57,64], and in An. gambiae sampled outdoors in northern Ghana [8].
Environmental conditions, including climate, have affected the distribution and species composition of malaria mosquitoes in the three main ecological zones of Ghana included in this study [39]. In the coastal savannah zone, An. melas thrive due to their tolerance of high salinity [65,66], whilst in the Sahel savannah zone some An. arabiensis are present due to the dry conditions (low Table 5 Frequencies of ace1 mutation of Anopheles gambiae s.l. at different sites in three ecological zones of Ghana For abbreviations, see Table 3 Site An important limitation of this study was not documenting the level of use of LLINs in each study site to better understand the changes in the resting behavior of the malaria vectors. However, the coverage of LLINs exceeds 70% in the study sites [70]

Conclusions
This study revealed high densities of An. coluzzii and An. gambiae with low genotypic insecticide resistance resting outdoors compared to indoors, which may have been triggered by current insecticide-based indoor interventions. This behavioral change in mosquito vectors could promote the outdoor transmission of malaria, since current control strategies mainly